Analyze the Data 3-3: Protein Purification The purification of a microtubule mot
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Question
Analyze the Data 3-3: Protein Purification
The purification of a microtubule motor protein has been undertaken (see Vale et al., 1985, Cell 42:39–50). A partially purified cell fraction containing the motor protein was subjected to an additional purification step using gel filtration chromatography. Aliquots of each fraction eluted from the gel filtration column were subjected to SDS gel electrophoresis. Fractions 21 to 41 are shown on the gel below, which was stained with Coomassie blue.
Fractions 28–32 from the gel filtration column were pooled and eluted over an ion-exchange
chromatography. Again, fractions were collected and aliquots of each were run on an SDS
polyacrylamide gel that was stained with Coomassie blue, as shown below. What can be
deduced about the relationship between the polypeptide that migrates at about 116 kD on the
gel and those that migrate at approximately 66–70kD?
205- 116- 66- 45- 21 23 25 27 29 31 33 37 41 Fraction numberExplanation / Answer
First lets understand wat Microtubule motor protein are and its composition.
Kinesin and dynein are the prototypes of microtubule motor proteins which move along microtubules in opposite direction. kinesin toward the plus end and dynein toward the minus end of microtubule.
Kinesin is a molecule of approximately 380 kd, consisting of two heavy chains (120 kd each) and two light chains (64 kd each)
Dynein is an extremely large molecule (up to 2000 kd), which consists of two or three heavy chains (each about 500 kd) complexed with a variable number of light and intermediate polypeptides, which range from 14 to 120 kd.
Ion exchange chromatography: based on this principle proteins are seperated dependent on their charged groupswhich causes the molecules to interact electrostatically with opposite charges on the stationary phase matrix.
Remember the molecules which are not attached to the column matrix are washed first. Hence the fractions obtained from 21 to 26 are washed first. In SDS PAGE (peptides move from positive end to negative end) these fractions have slow movement and have high molecular weight.
The peptide bands at 66-70 kd has the the same weight as two dynin light chain peptides.
The band at 116 resembles the heavy chain of kinesin peptides. which have molecular weight of 120kd.(heavy chains) The upper band might be the dynin protein of high molecular weight.